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See detailEffect of SK&F 96365 on extracellular Ca2+ -dependent O2- production in neutrophil-like HL-60 cells
Gallois, A.; Bueb, Jean-Luc UL; Tschirhart, Eric UL

in European Journal of Pharmacology (1999), 361(2-3), 293-8

Store-operated Ca2+ entry is referred to a capacitative current activated by Ca2+ -stores depletion in various non-excitable cells. Neutrophil-like HL-60 cells responded to N-formyl-L-Methionyl-L-Leucyl-L ... [more ▼]

Store-operated Ca2+ entry is referred to a capacitative current activated by Ca2+ -stores depletion in various non-excitable cells. Neutrophil-like HL-60 cells responded to N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP) by an early O2- production preceded by a [Ca2+]i rise. Cell stimulation in the absence of extracellular Ca2+ resulted in a major reduction of [Ca2+]i rise and O2- production. A purported inhibitor of store-operated Ca2+ entry, SK&F 96365 (1-(beta-(3-(4-methoxy-phenyl)propoxyl)-4-methoxy-phenetyl)- 1H-imidazole hydrochloride), inhibited extracellular Ca2+ -dependent [Ca2+]i rise by 30% but did not alter O2- production. In conclusion, SK&F 96365 did not modify extracellular Ca2+ -dependent O2- production, despite a significant but limited reduction in fMLP-activated membrane Ca2+ fluxes which can be ascribed to store-operated Ca2+ entry. Furthermore, Ca2+ influx is necessary for a full induction and maintenance of the biological response. [less ▲]

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See detailHuman umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5 respond to fMLP with [Ca2+]i variation and O2- production
Zardini, D. M.; Heuschling, Paul UL; Gallois, A. et al

in Journal of Immunological Methods (1997), 205(1), 1-9

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing ... [more ▼]

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. O2- production and [Ca2+]i rise were measured in these in vitro differentiated eosinophils after fMLP stimulation; with dihydrorhodamine-123 and fura-2, respectively. Umbilical cord blood-derived eosinophils responded to fMLP (0.01 nM to 3 microM) with a concentration-dependent production of O2- (EC50 = 63.1 +/- 17.2 nM; Emax = 71.0 +/- 6.2 pmol/min/10(6) cells). O2- production was correlated with an fMLP concentration-dependent increase in [Ca2+]i (EC50 = 32.5 +/- 14.9 nM; Emax = 200.0 +/- 23.9 nM). These results indicate that human umbilical cord blood-derived eosinophils demonstrate functional characteristics similar to adult human peripheral blood eosinophils after activation by fMLP. Therefore, the large numbers of eosinophils (2-3 x 10(6)/ml cord blood) which can be obtained by culture of human cord blood mononuclear cells may serve as a useful model for future studies which will provide insight into the pathogenesis of diseases associated with eosinophils. [less ▲]

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See detailEndothelin-1 does not modulate O2.release and [Ca(2+)]i variations in resting or differentiated HL-60 cells
Gallois, A.; Bueb, Jean-Luc UL; Tschirhart, Eric UL

in Fundamental & Clinical Pharmacology (1996), 10(1), 28-32

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O2.) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01-100 nM) was not able to ... [more ▼]

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O2.) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01-100 nM) was not able to modulate O2. generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC50 = 4.24 +/- 1.63 nM in the absence and 3.16 +/- 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01-100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca(2+)]i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O2.) or biochemical ([Ca(2+)]i response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O2. generation or [Ca(2+)]i variations. [less ▲]

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See detailA double-labelling fluorescent assay for concomitant measurements of [Ca2+]i and O2. production in human macrophages
Bueb, Jean-Luc UL; Gallois, A.; Schneider, J. C. et al

in Biochimica et Biophysica Acta (1995), 1244(1), 79-84

To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye ... [more ▼]

To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye dihydrorhodamine-123, which becomes fluorescent in its oxidized form, rhodamine-123. We describe a new double-dye technique whereby the kinetics of both [Ca2+]i levels and O2. production can be monitored simultaneously. This technique was developed in the dimethylsulfoxide-differentiated monocytic-like U-937 cell line (not equal to U-937), validated by comparison with single dye measurements and applied to human alveolar macrophages. The chemotactic peptide N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine induced in both cell types a similar transient elevation in [Ca2+]i, followed within seconds by a sustained increase in O2 production, which was however 4-fold weaker in not equal to U-937 cells. These results indicate that O2 production is an early event following the stimulation of human alveolar macrophages. This new double-dye technique may be relevant to other O2 ion-producing cells and could help to define more precisely the kinetics of the events leading to this biological response. [less ▲]

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